The human olfactory system is thought to detect thousands of odorants, allowing for proper food consumption and aversions. The ability to identify odors by humans is dependent on odorant receptors within the olfactory neurons that line the nasal epithelium. Odorant receptors (ORs) are clonally expressed in mature olfactory neurons (one OR allele per neuron). Thus studying odorant responses in identifiable individual neurons is equivalent to studying individual ORs. The human olfactory genome consists of ~900 OR genes;these genes belong to the seven-transmembrane receptor superfamily. However, only ~40% encode a full-length open reading frame (ORF). Thus, ~355 OR genes are available for odor identification in the main olfactory system. This subgroup of ORs breaks down into three OR Classes: Class I: ~50 genes;Class II: ~300 genes;and the TAARs: ~5 genes. With the emergence of SNP analysis, the number of variant, human ORs with full-length ORFs continues to grow. The entire set of Class I and II OR genes have been known for a decade, yet their ability to function in vivo has remained elusive. Three things are severely lacking in order to understand the functionality of all ~355 OR genes plus their variants: 1st) knowledge of which ORs are expressed in human olfactory neurons 2nd) evidence that the ORFs can couple to the olfactory G- protein (Golf) in an in vivo setting and 3rd) evidence that the ORFs can allow for glomerular formation in an in vivo setting. Failure for some human OR ORFs to drive glomerular formation or couple to Golf would classify these genes as "pseudogenes". Thus ORs having a full-length ORF does not constitute a valid enough reason for studying their odorant binding properties using in vitro technologies. There needs to be a method for quickly identifying which human OR ORFs can function in vivo and simultaneously make use of this in vivo system for the identification of odorant-OR activity correlates. In Aim 1 section A, a high-throughput gene-targeting strategy will be developed such that human OR ORFs can be rapidly "swapped" in place of a mouse OR ORF and be tested for functionality in vivo. Initial experiments will determine the efficacy of this approach at two mouse OR loci. The intent of this sub aim would be to generate a library of Embryonic Stem cells that each contain a unique human OR ORF. Initially, several isoforms for one human OR will be tested. In Aim 1 section B, a high-throughput transgenic approach will be developed whereby 70% of all olfactory neurons (millions) in the mouse olfactory epithelium will clonally express a given human OR. Again, several isoforms for one human OR will be tested. This project is aimed at complementing the gene- targeting strategy and providing a large number of neurons for possible robotic approaches in odorant identification. In short, the sole aim of this proposal is to move forward our understanding of which human receptors are functional for human perception of odors. PUBLIC HEALTH RELEVANCE: The ability for humans to detect odors (odorants) remains an important aspect of human health and welfare. The sense of smell is critically dependent on hundreds of different human odorant receptors normally expressed in olfactory neurons. However, very few odorant/odorant receptor matches have been made. This lack of knowledge is a sole reflection of the inability to express and characterize human odorant receptor proteins in non-olfactory cells. Thus, the functional analysis of human odorant receptors expressed within olfactory neurons should significantly help bridge this divide.